Ubiquinone is a generic term for 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone and is also called coenzyme Q. Ubiquinone is widely present in the biological world as a component of electron transfer systems. The polyprenyl side chain of ubiquinone has a different length depending upon the biological species and homologues of ubiquinone-1 to 13 have been found in nature. The main homologues are ubiquinone-6 to 10. Many mammals including humans biosynthesize ubiquinone-10.
Ubiquinone-10 is effective for the improvement of conditions involved in cardiac failure and other ischemic cardiac disorders and has been approved as a pharmaceutical. There has been a report that this substance is effective for reducing the cardiac side effects of anti-cancer agents such as Adriamycin, and for the improvement of periodontosis, and protection of skeletal muscles against load from exercise [Bitamin no Jiten (Dictionary of Vitamins), The Vitamin Society of Japan (1996)].
In recent years, activation of energy metabolism by ubiquinone and antioxidative effect of ubiquinone nave attracted attention and its demand as a healthy food has been expanded mainly in the U.S. and Europe.
Currently, ubiquinone-10 is produced by synthetic methods or through extraction from microorganisms such as yeasts and photosynthetic bacteria. However, a more efficient production process is required due to its increased demands.
One of the effective means to form and accumulate a specific substance using a microorganism is to block the flow of an intermediate metabolite on the biosynthetic pathway leading to a targeted product toward other pathways so that more of the intermediate metabolite flows to the targeted product.
Ubiquinone is structurally divided mainly into the quinone skeleton portion and the polyprenyl side chain portion. The polyprenyl side chain is a kind of isoprenoid containing 5-carbon isopentenyl pyrophosphate (IPP) as a basic skeletal unit and is biosynthesized by condensation of a plurality of IPP.
A series of enzymes participating in this reaction is called prenyltransferases.
Prenyltransferases have been found in many biological species. For example, in Escherichia coli, presence of three enzymes with different length of synthetic chain, farnesyltransferase [J. Biochem., 108, (6), 995-1000 (1990)], octaprenyltransferase [J. Bac., 179, 3058-3060 (1997)], and undecaprenyltransferase [J. Bac., 181, 483-492 (1999)], has been confirmed and the gene has been identified for all of them. In Rhodobacter sphaeroides (hereinafter referred to as R. sphaeroides) which is a photosynthetic bacterium, geranylgeranyl pyrophosphate synthetase (crtE) has been identified [J. Bac., 177, 2064-2073 (1995)].
The starting substrate for prenyltransferase that supplies ubiquinone side chain is considered to be farnesyl pyrophosphate (FPP) which is also the starting substrate for the biosyntheses of various isoprenoid.
In the case of R. sphaeroides, it is known that a remarkable amount of carotenoid accumulates from geranylgeranyl pyrophosphate (GGPP) which is formed by the action of crtE [Biosynthesis of Isoprenoid Compounds vol. 2, JOHN WILLY & SONS (1983)].
In the genera Pseudomonas and Rhodotorula, increased ubiquinone-10 accumulation has been reported by deletion of their carotenoid producibility (Japanese Published Unexamined Patent Application Nos. 68792/82, and 39790/82).
In R. sphaeroides, it has been already known that the carotenoid producibility disappears where crtE is defective, but there has been no finding that such defectiveness causes a change in the amount of intracellular accumulation of ubiquinone-10 [Mol. Microbiol., 4 977-989 (1990)].
In the above literature, mutants in which carotenoid biosynthetic ability is changed are obtained by the method wherein a mutagenic treatment is given to microorganism strains using radiations such as ultraviolet rays, X-rays, and γ-rays or chemicals such as sodium nitrite, nitrosoguanidine, and ethylmethyl sulfonate; strains showing a color change are selected from the mutated strains; and strains in which the ability to biosynthesize carotenoid is defective are further selected.
To readily cause a particular enzyme activity to become defective, a method of giving a mutation directly to a gene encoding the enzyme is also used. Various methods have so far been known. Among them, a method wherein targeted enzyme activity is inactivated by disrupting the gene encoding the enzyme by incorporating a vector containing a 5′- and 3′-terminals incomplete gene into the homologous region on chromosome and a method wherein DNA containing a gene that has lost its function by entire or partial deletion, substitution or insertion of the gene is used and the gene encoding the enzyme is disrupted by transferring the deletion, substitution or insertion on the chromosome to cause the targeted enzyme activity to become defective are known for their readiness and frequent use.
For the introduction of site-directed mutation into photosynthetic bacteria including R. sphaeroides, a method wherein a targeted gene is disrupted by conjugational transfer using a specific Escherichia coli and a vector is known. However, this method involves difficulties in that only limited vectors can be used and the separation process between Escherichia coli and a photosynthetic bacterium after conjugation is complicated. Construction of a site-directed homologous recombinant technique that is widely applicable regardless the kind of vectors is desired.
Another effective means to form and accumulate a specific substance using microorganisms is to strengthen the expression of an enzyme gene on the biosynthetic pathway.
In the case of ubiquinones, p-hydroxybenzoic acid biosynthesized via chorismic acid that is biosynthesized through the shikimic acid pathway is the starting substrate for the quinone skeleton portion. On the other hand, the starting substrate for the polyprenyl side chain portion is polyprenyl diphosphate formed by condensation of a plurality of IPP biosynthesized through the mevalonic acid pathway or through the recently clarified non-mevalonic acid pathway [Biochem. J., 295, 517 (1993)]. p-Hydroxybenzoic acid and polyprenyl diphosphate are converted to 4-hydroxy-3-polyprenylbenzoic acid by the action of p-hydroxybenzoic acid-polyprenyltransferase (EC.2.5.1.39), which undergoes various modifications to be converted to ubiquinone.
These enzymes on the biosynthetic pathways and their genes have been mostly identified in Escherichia coli and yeasts. Although the whole aspect of these genes have not yet been elucidated, several examples showing an increased accumulation of ubiquinone by strengthening the expression of enzyme genes on the biosynthetic pathway of ubiquinone are known. For example, Zhu et al. showed that the amount of ubiquinone accumulation increased by linking various enzyme genes of ubiquinone biosynthesis derived from Escherichia coli downstream to lac promoter and highly expressing them in Escherichia coli [J. Fermentation and Bioengineering, 79, 493 (1995)]. Also, Kawamukai et al. showed an improved productivity of ubiquinone-10 by introducing ubiA and ubiC derived from Escherichia coli into a photosynthetic bacterium, R. capsulatus, and carrying out culturing unaerobically (Japanese Published Unexamined Patent Application No. 107789/96)].
However, where the rate limiting step is in the biosynthesis of ubiquinone and what control does it undergo are yet to be elucidated.
To strengthen the biosynthetic system of ubiquinone in photosynthetic bacteria, it is considered to be most suitable to use enzyme genes of photosynthetic bacteria themselves. However, almost no enzyme gene that participates in the biosynthesis of ubiquinone is known with photosynthetic bacteria.
In order to strengthen the biosynthetic system of ubiquinone in photosynthetic bacteria, it is important to specify the rate limiting step on the biosynthetic pathway, isolate genes on the biosynthetic pathway of ubiquinone including the gene involved in the rate limiting step, and determine the nucleotide sequence of and around the genes.